Fluoroketone enzyme inhibitors

ABSTRACT

A method for enzyme immunoassay for a ligand suspected to be present in a liquid sample includes signal amplification by use of at least two enzymes and a blocked modulator for one of the enzymes. Ligand present in the liquid binds to an antiligand and an enzyme-labeled tracer. The resulting bound fraction is separated and the enzyme in the tracer removes the blocking group from the blocked modulator. The modulator activates or inhibits a second enzyme which catalyzes the conversion of a substrate to a product. The presence or absence of the ligand in the liquid is indicated by a signal, such as a color change or a rate of color change, associated with the product. The invention includes a new class of enzyme inhibitors and blocked inhibitors and a kit of materials useful for performing the method of the invention.

This is a division of application Ser. No. 07/282,816, filed Dec. 12,1988, abandoned; which is a division of application Ser. No. 932,951,filed Nov. 20, 1986, now U.S. Pat. No. 4,835,099.

FIELD OF THE INVENTION

This invention relates to immunoassay of an analyte and materials usedtherein, and more particularly relates to a method and materials forimmunoassay in which enhancement of a detectable signal is achieved bymodulation of enzymatic catalysis of an indicator reaction.

BACKGROUND OF THE INVENTION

Assay systems which are both rapid and sensitive have been developed todetermine the concentration of a substance in a fluid. Immunoassaysdepend on the binding of an antigen or hapten to a specific antibody andhave been particularly useful because they give high levels ofspecificity and sensitivity. These assays generally employ one of theabove reagents in labeled form, the labeled reagent often being referredto as the tracer. Immunoassay procedures may be carried out in solutionor on a solid support and may be either heterogeneous, requiring aseparation of bound tracer from free (unbound) tracer or homogeneous inwhich a separation step is not required.

Radioimmunoassay (RIA) procedures use radioisotopes as labels, providehigh levels of sensitivity and reproducibility, and are amenable toautomation for rapid processing of large numbers of samples. However,isotopes are costly, have relatively short shelf lives, requireexpensive and complex equipment, and extensive safety measures for theirhandling and disposal must be followed.

Fluoroimmunoassay (FIA) uses ftuorochromes as labels and provides directdetection of tile label. However, known homogeneous FIA methods usingorganic fluorochromes, such as fluorescein or rhodamine derivatives,have not achieved the high sensitivity of RIA, largely because of lightscattering by impurities suspended in the assay medium and by backgroundfluorescence emission from other fluorescent materials present in theassay medium.

Enzymes have also been used as labels in immunoassay. In conventionalenzyme immunoassay (EIA), an enzyme is covalently conjugated with onecomponent of a specifically binding antigen-antibody pair, and theresulting enzyme conjugate is reacted with a substrate to produce asignal which is detected and measured. When the signal is a colorchange, detection with the naked eye is limited because the averageindividual can detect the presence of chromophores only down to about10⁻⁵ or 10⁻⁶ M.

EIA sensitivity carl often be increased by spectrophotometrictechniques; however, these procedures require expensive equipment. Inanother approach, the sensitivity may be increased by variousamplification methods. Single enzyme amplification methods have beendisclosed in which ligands present at concentrations of 10⁻⁶ to 10⁻¹⁰ Mhave been detected. These methods however, have been generallyunsatisfactory at ligand concentrations above 10⁻¹¹ M. In cascadeamplification procedures, the number of detectable (generally colored)molecules is increased by use of two or more enzymes or enzymederivatives. U.S. Pat. No. 4,463,090 to Harris discloses a cascadeamplification immunoassay in which a large molecule activator, such asan enzyme or a proenzyme coupled to a ligand, activates a second enzymewhich reacts with a substrate to produce a detectable signal or in turnactivates a third enzyme.

U.S. Pat. No. 4,446,231 to Self discloses a cycling amplification enzymeimmunoassay which includes primary and secondary enzyme systems and amodulator for the second enzyme system. The primary system includes afirst enzyme coupled to a ligand. In a first embodiment of the Selfinvention, the first enzyme system acts on a modulator precursor toliberate a modulator. The modulator is a cofactor of the secondaryenzyme which activates the second enzyme system to catalyze the reactionof a substrate to a detectable product. During the reaction, themodulator is converted to an inactive form, and cycling is accomplishedby a third enzyme which reactivates the modulator. In a secondembodiment the modulator is an inhibitor of the secondary system, and isremoved by the primary enzyme system whereby the secondary system isactivated to act on the substrate and thereby produce the detectableproduct.

Boguslaski et al., U.S. Pat. No. 4,492,751 teaches a cycling system inwhich an enzyme substrate or coenzyme is conjugated to one member of thespecifically binding pair.

A variety of molecules has been shown to cause specific inactivation ofa target enzyme. A subset of inhibitors, termed mechanism-basedinhibitors, are substrates for enzymes which react with an enzyme toform a covalent bond. Mechanism-based inhibitors have been reviewed byWalsh (Tetrahedron 38, 871 (1982). Another subset of inhibitors includesmolecules which act as stable transition-state analogs. Gelb et al. havedisclosed some fluoroketones as transition-state inhibitors ofhydrolyric enzymes in Biochemistry 24, 1813 (1985).

SUMMARY OF THE INVENTION

One aspect of the present invention is a method for detection of aligand in a liquid sample, hereinafter referred to as the unknownsample. The unknown sample suspected of containing the ligand iscombined with a specific antiligand and a tracer for the ligand. Thetracer includes a first enzyme conjugated to either the ligand or to asecond antiligand, also specific for the ligand. Conditions conducive tobinding between ligand, antiligand and tracer are provided to give abound phase. After separation of the bound phase from the fluid, thebound phase is contacted in a liquid with a blocked modulator and asecond enzyme. The first enzyme removes the blocking group to provide amodulator for the second enzyme. A substrate for the second enzyme isthen added. The substrate is converted by the second enzyme to a productwhich provides a detectable signal, the conversion of the substrate tothe product by .the second enzyme being modulated by the modulator.

The ligand may be an antigen, a hapten or an antibody. The preferredligand is an antigen, most preferably a vital antigen. The method may becarried out by a competitive immunoassay technique, in which case thetracer is the ligand conjugated to the first enzyme. Preferably, asandwich immunoassay technique may be used, in which case the tracer isa second antiligand conjugated to the first enzyme.

The preferred blocked modulator of the present invention is a blockedinhibitor which is converted by the first enzyme component of the tracerto an inhibitor. The preferred substrate is a chromogen which isconvertible by the second enzyme to a product of a different color. Mostpreferably the chromogen is colorless and is converted to a coloredproduct by the second enzyme, the conversion of chromogen to productbeing inhibited by the inhibitor.

In the most preferred assay format, an antibody may be affixed to asolid support and contacted under binding conditions with a vitalantigen and a second antibody, specific for the antigen, labeled withalkaline phosphatase (first enzyme). After binding and separation, thesolid support having an antibody: antigen:alkaline phosphatase-labeledantibody sandwich affixed thereto is contacted with the blockedinhibitor and the second enzyme in a liquid. If antigen is present inthe fluid, alkaline phosphatase captured on the solid support removesthe blocking group to give the inhibitor. A colorless chromogen isadded. Inhibitor generated in the liquid inhibits the second enzyme fromconverting the chromogen to the -colored product. Thus the developmentof color indicates the absence of antigen in the sample and failure ofcolor development indicates the presence of antigen in the sample.

In another aspect of the invention, there is provided a new class ofenzyme modulators and blocked modulators. The preferred modulators areenzyme inhibitors, most preferably fluoroketones having a functionalgroup to which there is chemically bonded a blocking group which may becleaved by the first enzyme.

Another aspect of the invention includes a kit of materials forperforming the method of the invention substantially as described above.

In any EIA system which includes unblocking of an enzyme inhibitor,severe constraints are placed on both the inhibitor and the blockedinhibitor if the assay is to achieve maximum sensitivity. In accordancewith the present invention, the blocked inhibitor is an excellentsubstrate for the first (unblocking) enzyme, but is essentiallyunreactive toward the second (color forming) enzyme. Likewise, theunblocked inhibitor is a potent inhibitor of the second enzyme, but hasessentially no effect on the first enzyme. Because the reactions of theblocked inhibitor and inhibitor with the first and second enzymesrespectively are highly selective, "short circuits" characteristic ofprior art cycling EIA systems due to cross reactivities aresubstantially eliminated.

Thus, the invention provides a versatile method for assay for ligandspresent in very low concentrations in a fluid. The method makes possiblenaked eye detection and measurement of an assay signal even though theligand is present in concentrations as low as 10⁻¹² M and greatlyextends the range of ligands which can be detected or determined withoutexpensive or cumbersome equipment. Further, the assay sensitivity, i.e.,time required to detect the presence or absence of the ligand, may bereduced by up to 100 fold compared to conventional ElA. Significantsavings in cost and space are thereby achieved, enabling assays inaccordance with the invention to be carried out in small clinicallaboratories or even in a physician's office.

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE depicts the result of a typical assay in accordance with themethod and materials of the Invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

While this invention is satisfied by embodiments in many differentforms, there is described in detail preferred embodiments of theinvention, with the understanding that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the embodiments described. The scopeof the invention will be measured by the appended claims and theirequivalents.

In accordance with the method of the invention, a substance, hereinafterreferred to as the ligand, present in the unknown sample, may bedetected visually, i.e., by naked eye observation, even when present invery low concentrations. The method includes at least two amplificationstages. A first amplification stage is enzymatic unblocking of amodulator. A second amplification stage is enzymatic catalysis of anindicator reaction. These amplification steps take place sequentially toprovide signal amplification of 10⁶ fold or higher whereby a ligandpresent in the sample at a level of 10⁻¹² M or lower may be detectedwith the naked eye. If additional amplification is desired, a firstenzyme may be provided which initiates a cascade of sequential reactionsinvolving a plurality of enzymes, wherein any one or all of thereactions may provide further signal amplification.

An immunological reaction is used in the method of the invention fordetection of the ligand in the unknown sample. By the term"immunological reaction," as used herein, is meant a specific bindingreaction of an antigen and an antibody, a hapten and an antibody, or anyappropriate analogue of an antigen, an antibody, or a hapten which alsobinds specifically.

The immunological reaction may be carried out in any suitable liquid.For example, the liquid may be a body fluid suspected of containing theligand, such as serum, urine, cerebrospinal fluid, pleural fluid or thelike. Alternatively, the liquid may be water, saline or any appropriatebuffer, or a mixture of body fluids and other liquids to which has beenadded a sample suspected of containing ligand.

The preferred method for sandwich assay of the invention will first bedescribed with reference to the assay flow sheet below to provide ageneral understanding of the assay components and their interaction,after which each component will be discussed in detail. ##STR1## In theabove flow sheet, the following definitions apply, wherein a colonindicates an immunological binding, a hyphen indicates a chemical bondor a physical attachment, such as absorption, a solid arrow indicates achemical conversation and a dotted arrow indicates modulation of areaction or an assay component.

AL--antiligand

L--ligand

T--tracer

E₁ --first enzyme

E₂ --second enzyme

B-M--blocked modulator

M--modulator for enzyme

B--blocking group for modulator

Sub--substrate

P--product

It is seen from the flow sheet that conditions conducive to binding ofligand to antiligand and tracer are provided, followed by a separationstep and appropriate wash steps. A second enzyme and a blocked modulatorconsisting of a modulator (activator or inhibitor) and a blocking groupare added, and the blocking group is removed by the first enzymecomponent of the tracer. A substrate for the second enzyme is added, andthe conversion of the substrate to a detectable product by the secondenzyme is modulated by the modulator liberated from the blockedmodulator by the first enzyme. The level of product is either directlyor inversely proportional to the level of modulator, and thus to theligand, depending on whether the modulator is an activator or inhibitorrespectively. The actual signal measured may be a color associated withthe indicator reaction, as, for example, the color of the product orrate of formation thereof, or the color of the substrate or the rate ofdisappearance thereof.

In a preferred embodiment of the invention, one or more assay componentsmay be attached to the surface of a solid support. As known in the art,the solid .support may be any support which does not substantiallyinterfere with the assay. Exemplary of solid supports which may be usedare glass and polymeric materials, such as polyethylene, polyvinylidenefluoride, polystyrene and the like. Such supports may be fabricated intoany suitable shape, such as sheets, tubes, wells, or preferably, platessuch as microtiter plates. For example, an assay component may beattached to the inside walls and bottom of a tube, preferably a plastictube with one closed end, or most preferably, to the wells of amicrotiter plate. Preferably, after the desired quantity of antiligandis attached to the solid support, any remaining binding sites on thesupport may be filled by an inert protein. The inert protein may be anyprotein, as, for example, ovalbumin, which can be attached to thesupport and which does not interfere in any way with the specificbinding reactions between the ligand, antiligand and tracer, asdescribed below.

Preferably, the antiligand attached to the solid support is incubatedwith ligand and tracer to bind both to the solid support. After a washstep to remove interfering materials, the remaining assay components maybe added and the assay carried to completion as described below.

Turning now to a detailed description of the assay components, theligand may be from any source, and may be an antigen, an antibody or ahapten. For example, the ligand may be an antigen present in a bodyfluid, or it may be isolated from a body fluid and subsequentlyintroduced into a different liquid, such as buffer. In other cases, theligand may be from a source other than a body fluid, as, for example, aculture of microorganisms or a cellular extract thereof. Preferredligands are antigens, most preferably vital antigens present in a bodyfluid, such as Herpes simplex virus (HSV), Adenovirus, Influenza Avirus, parainfluenza 3 virus and Respiratory syncytial virus.

The antiligand is contacted with the ligand in the liquid to induce theimmunological reaction. The antiligand may be an antigen or an antibody,either monoclonal or polyclonal, or it may be any appropriate analoguethereof which reacts specifically with the ligand. In addition, theantiligand may be an antibody complex consisting of a plurality of boundantibodies, as, for example, a second antibody bound specifically to afirst antibody. Alternatively, the ligand may bind to several differentantiligands, for example, an ensemble of polyclonal antibodies or amixture of several monoclonal antibody molecules which bindsimultaneously to different surface areas of the ligand. Generally, thesecond antibody is raised against the first antibody in a differentspecies. The plurality of bound antibodies in the complex may containfrom about two to ten or more antibodies.

In the sandwich assay of the invention, it is preferred to use excessantiligand having sufficient binding sites to bind essentially all ofthe ligand.

The tracer comprises two components, the first enzyme conjugated to theligand (competitive assay as described later) or, in the preferredsandwich assay, to a second antiligand. The enzyme may be conjugated tothe ligand or antiligand in any suitable way, preferably by a covalentlinkage, prior to the immunological reaction. Covalent conjugation ofenzymes to ligands or antiligands is conventional and well known tothose skilled in the art.

Any enzyme which can remove a blocking group from a blocked modulatormay be used as the first enzyme. Suitable first enzymes are generallyhydrolases, such as phosphatases, peptidases, esterases, glycosidasesand the like. Exemplary of, but not limited to, suitable first enzymesare trypsin, thrombin, mammalian liver esterase, acetylcholinesterase,β-galactosidase, or most preferably, alkaline phosphatase.

The liquid containing the ligand, the antiligand and the tracer may beincubated, if necessary, to induce binding. Incubation may be carriedout at any temperature and for any length of time suitable to facilitatebinding, preferably from about 20° to 40° for about 1 minute to 4 hours.Antiligand, ligand and tracer which are bound are hereinafter referredto as the bound fraction and antiligand, ligand and tracer which do notbind are hereinafter referred to as the free fraction. The assay may,but need not, be carried out in such a way that equilibrium isestablished between the bound and free fractions.

The bound fraction may be separated from the free fraction in the liquidphase of the assay mixture by any conventional method, such asfiltration, decantation, centrifugation, aspiration and the like. Whenthe immunological reaction has been carried out on a solid support, theliquid phase is conveniently decanted and the solid support washed toensure removal of the free fraction and any other materials which wouldinterfere with the assay and resuspended in a suitable liquid such aswater, saline or buffer. The blocked modulator is then added, and the pHis adjusted to any level, preferably 6-8, which does not causenon-enzymatic removal of the blocking group, as described below.

The blocked modulator may be any material which may be converted by thefirst enzyme to a modulator of the second enzyme. The preferred blockedmodulator has two components, the modulator and the blocking group. Themost preferred blocked modulator is a blocked inhibitor which isunreactive toward the second enzyme until its blocking group is removedby the first enzyme and the inhibitor is liberated into the assaymedium. Thus, the choice of the components of the blocked inhibitordepends on the first and second enzymes to be used. The blocking groupshould be one which can be covalently conjugated to the inhibitor by abond which can be cleaved substantially selectively by the first enzyme,and the inhibitor component should inhibit the activity of the secondenzyme while having substantially no effect on the first enzyme. Thus,the nature of the second enzyme and its substrate will be discussedprior to further description of the blocked inhibitor and the inhibitor.

In the assay of the invention, the second enzyme is generally ahydrolase which converts the substrate to the product. Suitablehydrolases are, for example, phosphatases, peptidases such as trypsin,chymotrypsin and pepsin, or preferably esterases such as acetylcholinesterase (ACHE) and butyl cholinesterase. The most preferredsecond enzyme is a carboxyesterase, such as rabbit liver esterase (RLE).

The substrate may be any substance containing a group which can becleaved by the second enzyme to provide a product detectable by a signalassociated with color. Thus, in one embodiment of the invention, thesignal detected is the development or disappearance of a color, or achange from one color to another. In another embodiment of theinvention, the signal may be a change in the rate at which the substrateis converted to the product, for example, the color of a substrate maybe observed to remain unchanged for a specified length of time. Thus,measurements of the signal may be made under either kinetic orthermodynamic conditions. Kinetic measurements determine the rate ofchange which occurs over a period of time, and are generally carried outby making a series of measurements at various times after combining theassay reagents. Thermodynamic measurements determine the extent ofchange which has occurred when equilibrium has been reached between thesubstrate and the product of the indicator reaction. Measurements may.be made either instrumentally or, preferably, with the naked eye.

It is preferred that the substrate be colorless until cleaved by thesecond enzyme to give a colored product. Suitable substrates are indoxylesters and, preferably, esters of nitrophenols, such as ortho and paranitrophenyl acetates or buryrates. These substrates are colorless untilcleavage of the acetyl or butyryl groups by carboxyesterase occurs togive colored nitrophenols. Thus, when the modulator is an inhibitor andthe substrate is an ester of a nitrophenol, the signal which is measuredis inhibition of color formation.

It is evident that the method of the invention may also be used in afluorescence immunoassay. In this embodiment of the invention, thesecond enzyme may convert a nonfluorogenic substrate to a fluorogenicproduct wherein the signal measured is modulation of fluorescence. Forthis embodiment of the invention, it is preferred that the modulator bean inhibitor and the second enzyme be an esterase.

As mentioned above, the first enzyme component of the tracer cleaves theblocking group from the blocked inhibitor to provide the inhibitor ofthe second enzyme, and another aspect of the invention is a new class ofenzyme inhibitors and blocked enzyme inhibitors of the general formulasI-IV, set forth below, wherein the nature of group B, as describedlater, determines whether the compound is an inhibitor or a blockedinhibitor: ##STR2##

In formulas I-IV, R₁ may be H, lower alkyl of 1-6 carbon atoms, branchedor unbranched, or ##STR3## wherein R₂ may be lower alkyl of 1-6 carbonatoms; R₃ may be H, nitro, alkoxy, halogen and the like; R₄ may be analkyl group of 1-10 carbon atoms or an alkenyl or alkynyl group of 2-10carbon atoms optionally substituted with an aryl group or an aryl groupsubstituted with a nitro, hydroxyl, mercapto, alkyloxy, haloalkyl,hydroxyalkyl, mercaptoalkyl group; Y and Z may independently be H or Fwherein at least one of Y and Z is F; X may be O, S or NR₅ wherein R₅may be H or R_(2;) n may be 1-6; m may be 2-6; A may be F or CF3; and Bmay be H, a phosphoric acid or salt, a glycosyl group, an amino acidresidue, such as a lysine or arginine residue covalently conjugated to Xthrough the amino acid carboxyl group, an acyl group of 2-4 carbon atomssuch as an acetyl or butyryl group, or a peptide of the formula ##STR4##R₇ may be H or lower alkyl of 1 to 6 carbon atoms, branched orunbranched; R₈ may be H, lower alkyl or hydroxy-lower alkyl of 1 to 4carbon atoms, branched or unbranched, CH₂ COOH or (CH₂)₂ COOH; R₉ may belower alkyl or lower alkoxy of 1 to 4 carbon atoms, branched orunbranched, phenyl, or benzyloxy; and q may be 0-10.

When B is H, formulas I to IV represent enzyme inhibitors. When B is anygroup other than H, formulas I to IV represent blocked enzymeinhibitors. When B is a phosphoric acid or salt thereof, it is intendedthat B have the formula ##STR5## wherein P is bonded to X and n may beas described above.

The inhibitor and blocked inhibitor in accordance with formulas I to IVmay be synthesized by any sequence of conventional chemical reactions asmay be envisioned by one skilled in the art. Suitable and convenientmethods are given in the Examples, below. The following list ofeffective enzyme inhibitors is intended to be exemplary only.

    __________________________________________________________________________                                            Ki (M).sup.                           Name               nmr data             (Esterase)                            __________________________________________________________________________      1,1,1-trifluoro-3-(4-hydroxy-                                                                  (CDC13) - 3.91(s, 2H), 5.21(bs, 1H),                                                               2.0 × 10-6, RLE                   phenyl)propanone 6.90(d, 2H), 7.10(d, 2H)                                     1,1,1-trifluoro-3-(3-hydroxy-                                                                  (CDC13) - 4.00(s, 2H), 4.80(bs, 1H),                                                               >10-4, PLE                              phenyl)-2-propanone                                                                            6.80(m, 3H), 7.30(m, 1H)                                     1,1,1-trifluoro-4-(4-hydroxy-                                                                  (CDC13) - 2.95(m, 4H), 4.90(bs, 1H),                                                               2.0 × 10-8, RLE                   phenyl)-2-butanone                                                                             6.92(dd, 4H)J=4,60Hz                                         1,1,1-trifluoro-4-(3-hydroxy-                                                                  (CDC13) - 2.94(t, 2H), 3.05(t, 2H),                                                                1.0 × 10-7, RLE                   phenyl)-2-butanone                                                                             5.70(bs, 1H), 6.80(m, 3H), 7.15(m1H)                         1,1,1-trifluoro-5-(4-hydroxy-                                                                  (CDC13) - 1.91(t, 2H), 2.59(t, 2H),                                                                1.0 × 10-8, RLE                   phenyl)-2-pentanone                                                                            2.68(t, 2H), 5.23(bs, 1H), 6.95(d, 2H),                                       7.10(d, 2H)                                                  1,1,1-trifluoro-5-(3-hydroxy-                                                                  (CDC13) - 1.95(p, 2H), 2.70(t, 2H),                                                                1.7 × 10-7, RLE                   phenyl)-2-pentanone                                                                            2.95(t, 2H), 5.40(bs, 1H), 6.70(m, 3H),                                       7.30(m, 1H)                                                  1,1,1-trifluoro-6-(4-hydroxy-                                                                  (CDC13) - 1.63(m, 4H), 2.59(q, 2H),                                                                2.0 × 10-8, RLE                   phenyl)-2-hexanone                                                                             2.70(q, 2H), 5.55(bs, 1H), 6.77(d, 2H)                                        7.02(d, 2H)                                                  1-phenyl-3,3-difluoro-10-                                                                      (CDC13) - 1.35(m, 4H), 1.65(m, 4H),                                                                --                                      hydroxy-4-decanone                                                                             2.25(m, 2H), 2.70(q, 2H), 2.75(t, 2H),                                        3.15(bs, 1H), 3.55(t, 2H), 7.25(m, 5H)                       1,1,1-trifluoro-5-hydroxy-                                                                     (CDC13) - 2.15(m, 4H), 4.03(bs, 1H),                                                               3.0 × 10-4, PLE                   2-pentanone      4.20(m, 2H)                                                10.                                                                             1,1,1-trifluoro-6-hydroxy-                                                                     (CDC13) - 1.85(m, 4H), 2.10(bs, 1H),                                                               4.0 × 10-7, PLE                   2-hexanone       2.30(m, 4H)                                                  1,1,1-trifluoro-8-hydroxy-                                                                     (CDC13) - 1.30-1.80(m, 8H), 2.40                                                                   --                                      2-octanone       (bs, 1H), 2.75(t, 2H), 3.65(m, 2H)                           1-hydroxy-5,5-difluoro-8,8-                                                                    (CDC13) - 0.95(s, 9H), 1.45(t, 2H),                                                                1.2 × 10-6, PLE                   dimethyl-4-nonanone                                                                            2.00(m, 6H), 3.12(bs, 1H), 4.15(m, 2H)                       1,1,1,2,2-pentafluoro-5-(4-                                                                    (CDC13) - 2.94(m, 2H), 3.04(m, 2H),                                                                8.0 × 10-7, RLE                   hydroxy-phenyl)-3-pentanone                                                                    4.75(bs, 1H), 6.90(d, 2H), 7.10(m, 2H)                       1,1,1-trifluoro-4-(3-hydroxy-                                                                  (CDC13) - 5.90(bs, 1H), 6.90(d, 1H)                                                                1.6 × 10-7, RLE                   phenyl)-3-trans-buten-2-one                                                                    j=16HZ, 7.25(m, 4H), 7.95(d, 1H) j=16Hz                      N,N-dimethyl-N-[2-(hydroxy)ethyl]                                                              (D20) - 1.90(m, 4H), 3.13(s, 6H),                                                                  5.0 × 10-9, AChE                  5,5,5-trifluoro-4-oxopentan-                                                                   3.30(m, 2H), 3.48(m, 2H), 4.01(bs, 2H).                      aminium, hydroxide salt                                                       N,N-dimethyl-N-[4-(hydroxy)butyl]                                                              (D20) - 1.95(m, 4H), 2.54(m, 4H)                                                                   6.5 × 10-8, ACHE                  5,5,5-trifluoro-4-oxopentanaminium,                                                            3.10(s, 6H), 3.35(m, 2H), 3.55(m, 2H),                       hydroxide salt   5.25(bs, 1H)                                               __________________________________________________________________________     .sup. PLE, Pig Liver Esterase (E.C. 3.1.1.1)                                  RLE, Rabbit Liver Esterase (E.C. 3.1.1.1)                                     AChE, Acetyl Choline Esterase (E.C. 3.1.1.7)                             

In the most preferred embodiment of the invention, the tracer is asecond antiligand having alkaline phosphatase covalently conjugatedthereto. After separation and washing of the solid support andresuspension thereof in a suitable assay liquid, RLE or AChE (secondenzyme) and the blocked inhibitor of formula V is added. If antigen ispresent in the unknown fluid, it and the tracer are captured on thesupport, and the alkaline phosphatase causes cleavage of the phosphateester bond of V to give inhibitor of formula VI. The assay is completedby addition of o-nitrophenylbutyrate or o-nitrophenylacetate, VII.##STR6##

If inhibitor VI formed due to the presence of antigen in the unknownfluid, the activity of the esterase is inhibited, and colorlesssubstrate VII is not converted to colored product VIII. If no antigen ispresent in the unknown fluid, no alkaline phosphatase is captured on thesolid support, no inhibitor VI is formed and colored product VIIItherefore forms because the esterase is not inhibited.

In this most preferred embodiment of the invention using 1×10⁻¹² Malkaline phosphatase and 3×10⁻⁹ M RLE as first and second enzymesrespectively, the presence or absence of an antigen in the unknown fluiddown to a level of 1×10⁻¹² M can be detected in about seven minutes. Ifantigen is present in the unknown fluid, no color detectable with thenaked eye (ca 1×10⁻⁵ M of product VIII) develops in this time. If noantigen is present, color does develop. In contrast, by conventionalimmunoassay using first enzyme (alkaline phosphatase) alone, about 104minutes are required for the enzyme to cleave sufficient phosphorylatednitrophenol to provide detectable color. Thus, the invention representsa 15 fold increase in assay sensitivity using these reagents at theselevels.

As mentioned earlier, another embodiment of the invention is acompetitive assay wherein the tracer is a predetermined quantity of theligand conjugated to the first enzyme. In a competitive assay, thequantity of antiligand used is insufficient to bind all of the ligandand tracer present in the assay liquid so that ligand and tracer competefor the limited number of antiligand binding sites. Thus, in acompetitive assay, the quantities of ligand and tracer which bind to theantiligand (bound fraction) are inversely proportioned to theirconcentrations in the assay liquid.

If additional signal amplification is desired, a multistage cascadeamplification assay may be carried out wherein a plurality of reagentsin the assay medium react sequentially leading ultimately to modulatorunblocking. In describing this embodiment of the invention, it isconvenient to consider the first enzyme described above as a primaryenzyme which enzymatically converts a reagent in the assay medium to asecondary enzyme which unblocks the modulator for the above-describedsecond enzyme. Further, the secondary enzyme, or any subsequent enzyme,may react with additional reagents to provide additional enzymes whichmay continue the cascade of enzymatic reactions until the modulator isunblocked. By proper selection of reagents to be added to the assaymedium, any desired number of amplification stages may be carried out.

Amplification occurs in any embodiment of the invention heretoforedescribed because the first enzyme, or any subsequently formed enzyme,and the second enzyme act as true catalysts wherein a single enzymemolecule may act on an essentially unlimited number of blocked modulatoror substrate molecules respectively without being consumed. Thus, intheory, one molecule of each enzyme would be sufficient to perform themethod of the invention. In practice, determination of the amounts ofthe enzymes to be added End the number of amplification stages to beused depend on the level of amplification desired and are well withinthe purview of one of ordinary skill in the art.

It is evident that an almost unlimited number of competitive andsandwich assay configurations which fall within the scope of theinvention can be envisioned. Further, the invention provides assayconfigurations which are suitable for either detection of the ligand ordetermination of ligand concentration. Ligand concentration may bedetermined by comparing the magnitude of the signal generated with theunknown sample with the magnitude of the signal measured upon assay of arange of known quantities of the ligand assayed under essentiallyidentical conditions. When the method of the invention is to be used fordetermination of ligand concentration, it is advantageous to read signalintensity by an appropriate instrument, such as a spectrophotometer, forexample, a Beckman DU7 Spectrophotometer, Beckman Instruments, Inc.,Irvine, Calif.

In another embodiment of the invention, the second enzyme may be affixedto the solid phase in close proximity to the antiligand. After theimmunological reaction by which the ligand and tracer are captured onthe solid support, the support is separated from the liquid phase,washed, and tile blocked modulator is added. The first enzyme portion ofthe tracer reacts with and liberates the modulator for the second enzymeon the support. The substrate for the second enzyme may then be addedand the assay completed as previously described.

Another aspect of the invention is a reagent kit or package of materialsfor performing an assay for a ligand in accordance with the method ofthe invention. The kit may include one or more antiligands, a firstenzyme which may be conjugated to one of the antiligand, or to theligand, a second enzyme, and a blocked modulator for the second enzymewherein one of the antiligands may optionally be attached to a solidsupport. The kit may also include standards for the ligand, as, forexample, one or more ligand samples of known concentration, or it mayinclude other reagents, enzyme substrates, or other labeled or unlabeledspecific ligands, antiligands or complexes thereof useful in carryingout the assay. It may include solutions, such as saline or buffers. Thecomponents of the kit may be supplied in separate containers, as, forexample, vials, or two or more of the components may be combined in asingle container.

EXPERIMENTAL

Routine Analytical Techniques--Flash Silica gel chromatographywas-performed on ICN silica gel 32-63 mesh at 3-7 psi. Analytical TLCwas performed on 0.25 mm 5×20 cm aluminum-backed silica gel plates fromEM Scientific. Preparative TLC was performed on 2.0 mm 20×20 cmglass-back silica gel plates from EM Scientific. Melting points wereperformed on a Thomas Hoover capillary melting point apparatus and areuncorrected. NMR spectra were recorded on an IBM WP-200SYspectrophotometer and chemical shifts are reported in ppm relative totrimethylsilane. HPLC was performed on a Waters 510 two pump system withUV detection using one of two solvent systems on a Brownlee AX- 3007×250 mm column; System A) initial hold for 5 minutes at 30 mM NH4OAc pH6.5 followed by a linear gradient to 2.0 M NH4OAc over a 30 minuteperiod followed by a hold at 1.0 M NH4OAc for 5 minutes. System B) usedan isocratic buffer system of 30 mM NH4OAc pH 6.5 for 40 minutes. Flowrates were 1.0 mL/minute. Gas Chromatography was performed on a H.P.5840A Gas Chromatograph equipped with a FID and an automatic injectorusing a 30 M DB-1 Megabore column purchased from J&W Scientific, Inc. GCconditions were as follows; A three minute hold at 100° C. followed by a10° C./minute gradient to 250° C. followed by a 3.0 minute hold at 250°C. at 16.0 mL/minute flow rate.

Inhibition constants were measured in 50 mM Tris pH=8.0. Enzyme andinhibitor were incubated at ambient temperature for 20 minutes.Substrate for the enzyme was then added and the rate of hydrolysis wasfollowed spectrophotometrically. The substrate for PLE and RLE waso-nitro-phenylbutrate and for AChE was acetyl thiocholine and Ellman'sreagent.

EXAMPLE I Diammonium [4-(3-oxo-4,4,4-trifluorobutyl)phenyl]phosphate A.Ethyl 3-(4-methoxyphenyl)-2-(1-oxo-2,2,2-trifluoroethyl)propionate

A 1L four neck round bottom flask, fitted with reflux condenser,dropping funnel, argon inlet, and magnetic stirrer was charged with 7.17g (0.149M) of a 50% oil dispersion of sodium hydride and 300 mL of dryethyl ether. Nine mL of absolute ethanol was slowly added to thestirring solution. After the evolution of hydrogen stopped, a mixture of25 g(0.136M) of ethyl 4,4,4-trifluoromethyl acetoacetate and 21.3g(0.136M) of 4-methoxybenzyl chloride was added over a 1 hour period.The mixture was then heated at reflux overnight. The reaction mixturewas then cooled, extracted with water, 1N HCl, dried (anhydrous MgSO4),and solvent removed under reduced pressure. The crude reaction mixture,33.5 g, was chromatographed on a 60×300 mm silica gel column with a 1:3ethyl acetate:hexane mixture. Similar fractions were combined and gave9.0 g (27%) of the desired product as an oil. nmr(CDCl3) - L 2.12(m,3H),2.67(m,2H), 3.85(m,3H), 3.90(S,3H), 7.24(q,4H).

B. 1,1,1-Trifluoro-4-(4-hydroxyphenyl)-2-butanone

A 100 mL round bottom flask, fitted with reflux condenser, magneticstirrer and argon inlet was charged with 2.05 g(6.7mM) of ethyl3-(4-methoxyphenyl)-2-(1-oxo-2,2,2-trifluoroethyl)propionate, 20 mL of31% HBr in AcOH, and 10 mL of water. This mixture was heated overnightat 120° C., concentrated under reduced pressure and partitioned betweendichloromethane and water. The organic layer was extracted with aqueousbisulfite, saturated sodium bicarbonate, dried (anhydrous MgSO4), andthe solvent removed under reduced pressure. The crude reaction mixturewas chromatographed on a 50×300 mm silica gel column with 1:1 ethylacetate:hexane. Similar fractions were combined and solvent was removedunder reduced pressure to yield 600 mg(41%) as a clear oil. nmr(CDCl3) -L 2.95(m,4H), 4.90(bs,1H), 6.93(dd,4H)J=4,60Hz.

C. Diethyl [4-(3-oxo-4,4,4-trifluorobutyl)phenyl] phosphate

A 10 mL one neck round bottom flask, fitted with argon inlet andmagnetic stirrer was placed in an ice bath and charged with 400 mg(1.8mM) of 1,1,1-trifluoro-4-(4-hydroxyphenyl)-2-butanone, 400 mg(2.4 mM) ofdiethyl chlorophosphate, 0.15 mL of dry pyridine and 5 mL ofdichloromethane at 5° C. The mixture was stirred overnight at ambienttemperature, filtered to remove pyridine HCl, extracted with 0.2N HCl,extracted with water, and dried (anhydrous MgSO4). Solvent removal underreduced pressure gave a crude yield of 600 mg of a brown oil.Preparative TLC using 1:1 ethyl acetate:hexane gave 600 mg(92%) of aclear oil nmr(CDCl₃) - L 1.50(m,6H), 3.0(m,4H), 4.20(m,4H), 7.15(s,4H).

D. Diammonium [4-(3-oxo-4,4,4-trifluorobutyl)phenyl] phosphate

A 25 mL one neck round bottom flask, fitted with argon inlet andmagnetic stirrer was charged with 5.0 mL of dichloromethane, 140mg(0.4mM) of diethyl [4-(3-oxo-4,4,4-trifluorobutyl)phenyl] phosphate and 2.0mL of bromotrimethylsilane. After stirring this mixture for 3 hours atambient temperature, 10 mL of methanol was added and the volatilematerials were removed under reduced pressure. The residue was dissolvedin water and the pH adjusted to 7.3 with 1.0 N NaOH. The aqueoussolution was extracted with ethyl ether and lyophilized to give 190 mgof a white solid. This material was dissolved in 10 mL of 30 mM NH4OAcbuffer and purified by HPLC using system A. Yield of the product afterlyophilization was 50 mg (37%). mp 235°-240° C. nmr(D₂ O) -L 1.90(m,2H),2.56(m,2H), 4.65(s,DOH), 6.88(dd,4H)J=6, 82 Hz.

EXAMPLE II 1-Hydroxy-5,5-difluoro-8,8-dimethyl-4-nonanone A. Ethyl2,2-difluoro-5,5-dimethylhexanoate

A 100 mL three neck round bottom flask fitted with dropping funnel,argon inlet, ice bath, and magnetic stirrer was charged with 8.0 mL ofdichloromethane and 2.21 g(20 mM) of ethyl 2-oxo-5,5-dimethylhexanoate.Diethylaminosulfur trifluoride, 2.11 g(13 mM), in 5 ml ofdichloromethane was added to the reaction mixture over a 15 minuteperiod and the mixture was stirred overnight at ambient temperature. Thereaction mixture was partitioned between water and dichloromethane, theorganic layer dried (anhydrous MgSO4), and the solvent removed underreduced pressure. The oily residue was distilled at 83°-88° C. at 20 mmto give 1.0 g(25%). nmr(CDCl3) -L 0.95(s.9H), 1.40(m,.4H), 2.10(m,2H),4.35(q,2H).

B.2,3,4,5-Tetrahydro-2-oxo-3-[(5,5-dimethyl-2,2-difluoro-1-oxo)hexyl]furan

A 25 mL three neck round bottom flask, fitted with drying tube, droppingfunnel, heating mantle, and magnetic stirrer was charged with 0.24 g(5.0mM) of sodium hydride in a 50% oil dispersion. The sodium hydride waswashed with dry hexane (2×10 mL) and 5.0 mL of ethyl ether was added tothe flask. A mixture of 5 drops of absolute ethanol and 5 mL of etherwas slowly added to the sodium hydride suspension. After the evolutionof hydrogen had stopped, a mixture of 1.0 g(5.0 mM) of ethyl2,2-difluoro-5,5-dimethylhexanoate and 0.43 g(5.0 mM) of γ-butyrolactonein 5.0 mL of ethyl ether was added over a 20 minute period. The solutionwas refluxed for 3 hours and allowed to stir at ambient temperatureovernight. The reaction mixture was partitioned with 1N HCL, the organiclayer washed with water (2×50 mL), dried (anhydrous MgSO4), and thesolvent removed under reduced pressure. The oily residue, 0.88 g, wascrystallized from a hexane:ethyl acetate mixture, 0.66 g (53%). nmr -(CDCl₃) -L 0.95(s,9H), 1.35(m,2H), 2.00(m,3H), 2.50(m,2H), 3.00(mlH),4.25(m,1H), 4.5(m,1H).

C. 1-Hydroxy-5,5-difluoro-8,8-dimethyl-4-nonanone

A 10 mL one neck round bottom flask, fitted with argon inlet, magneticstirrer, and reflux condenser was charged with 1.0 mL glacial aceticacid, 4 drops of concentrated HCl and 200 mg(0.81 mM) of2,3,4,5-tetrahydro-2-oxo-3-(5,5-dimethyl-2,2-difluoro-1-oxohexyl)furan.The reaction mixture was heated at 110 C. overnight under a blanket ofargon. The reaction was extracted with ethyl ether, and the ether wascross washed with water, dried (anhydrous MgSO4), and solvent removedunder reduced pressure. The residue was chromatographed on a 10×60 mmsilica gel column using a 9:1 hexane:ethyl acetate mixture to give aclear oil. nmr(CDCl₃) -L 0.95(s,9H), 1.45(t,2H), 2.00(m,6H),3.12(bs,1H), 4.15(m,1H)

EXAMPLE IIIN,N-dimethyl-N-[2-(hydroxy)ethyl]-5,5,5-trifluoro-4-oxopentanaminium,hydroxide salt A.2,3,4,5-Tetrahydro-2-oxo-3-[(2,2,2-tri-fluoro-1,1-dihydroxy)ethyl]furan

A 3L three neck round bottom flask, fitted with reflux condenser,dropping funnel, argon inlet, magnetic stirrer, and heating mantle wascharged with 36 g(0.75M) of a 50% oil dispersion of sodium hydride. Thesodium hydride was washed (2×200 mL) with dry hexane and then suspendedin 800 mL of ethyl ether and 2 mL of absolute ethanol. A mixture of 60.2g(0.7M) of γ-butyrolactone and 99.4 g(0.7M) of ethyl trifluoroacetate in750 mL of ether was added to the suspension at a rate to keep thereaction at gentle reflux. The mixture was refluxed for an additionaltwo hours and then stirred at ambient temperature overnight. Thereaction was cooled in an ice bath and 1N HCl (250 mL) was added. Theorganic layer was separated, washed with water (2×200 mL), dried(anhydrous MgSO4), and solvent removed under reduced pressure. Theresidue was crystallized from hexane:ethyl acetate to give 64.0 g(45.4%), mp- 87-90

C. nmr(DMSO-d6) -L 2.33(m,2H), 3.09(t,1H)J=7Hz, 4.20(m,2h), 7.00(s,1H),7.52(s,1H). B. Preparation of 1,1,1-trifluoro-5-hydroxy-2-pentanone

A 500 mL three neck round bottom flask, fitted with reflux condenser,heating mantle, and magnetic stirrer was charged with 61.5 g(0.31M) of2,3,4,5-tetrahydro-2-oxo-3-(2,2,2-trifluoro-1,1-dihydroxyethyl)furan,6.4 mL of concentrated HCl, 10 mL of water, and 80 mL of acetic acid.The reaction mixture was heated at 125 C. overnight. An additional 3.0mL of concentrated HCl and 5.0 mL of water was added and the reactionmixture was heated for an additional 10 hour. The reaction waspartitioned between water and ethyl ether while the water layer wasneutralized with solid sodium bicarbonate (100 g). The ether solutionwas washed with water (2×20 mL), dried (anhydrous MgSO4), and solventremoved under reduced pressure to give 60 g(45%) of a pale yellow oil.nmr(CDCl₃)-L 2.15m,4H), 4.03(m,1H), 4.20(m,1H).

C. 1,1,1-Trifluoro-5-bromo-2-pentanone

A 500 mL three neck round bottom flask, fitted with dropping funnel,magnetic stirrer, ice-salt bath, and argon inlet is charged with 125 mLof dry dimethylfofmamide, 29 g(35.7 mM) of tributyl phosphine, and 11.2g(54.9 mM) of 1,1,1-trifluoro-5-hydroxy-2-pentanone. This mixture wascooled to -5 C. and 11.5 g(71.6 mM) of bromine was added dropwise over a2 hour period. After stirring overnight at ambient temperature thereaction-mixture was distilled though a 30 cm vigreaux column at 2.0 mmof pressure. Two fractions were collected; the first fraction from 27-35C. and the second fraction at 35-70 C. The second fraction waspartitioned between water and ethyl ether, the organic layer was washedwith water (3×100 mL), dried (anhydrous MgSO4), and evaporated underreduced pressure at ambient temperature to give 30 g of a colorless oilymixture of dimethyl formamide, ether, and the desired product which wasused in the next reaction without further purification.

D. N,N-dimethyl-5,5,5-trifluoro-4-oxopentanamine

A 500 mL three neck round bottom flask, fitted with dropping funnel,argon inlet, magnetic stirrer, and ice-salt bath was charged with 21.5g(0.45M) of anhydrous dimethylamine at -8 C. To this mixture was added19.4 g(88.5 mM) of 1,1,1-trifluoro-5-bromo-2-pentanone in dimethylformamide and ethyl ether dropwise at -10 C. The suspension was stirredfor 2.5 hours at -10 C. The solvent was then decanted from theprecipitate. The precipitate was washed with ethyl ether (3×200 mL), theorganic layers were combined, washed with water (2×30 mL), dried(anhydrous MgSO4), and evaporated under reduced pressure to give 15.0g(92%) of a pale yellow oil. nmr of HCl salt (CDCL₃) - 1.89(s,4H),2.90(s,6H), 3.21(t,2H).

E. N,N-dimethyl-N-[2-(methoxy)ethyl]-5,5,5-trifluoro-4-oxopentanaminium,hydroxide salt

A 25 mL one neck round bottom flask was charged with 2.95 g (21.2 mM) of2-bromoethyl methyl ether, 0.33 g(1.77 mM) ofN,N-dimethyl-5,5,5-trifuluoro-4-oxopentanamine) and 2.5 mL of dimethylformamide and the mixture was stirred at ambient temperature for 3 days.Solvent was removed under reduced pressure and the amber oil waschromatographed on a Dowex-1 (15×200 mm) column in the hydroxide formwith 200 mL of water. The effluent from the column was lyophilized toyield 0.28 g(60%) of an amber oil which was the as the hydroxide salt.nmr(D₂ O)-product 1.94(m,2H), 3.13(s,6H), 3.39(s,3H), 3.41(m,2H),3.60(m,2H), 3.88(bs,2H). The material was used in next reaction withoutfurther purification.

F. N,N-dimethyl-N-[2-(hydroxy)ethyl]-5,5,5-trifluoro-4-oxopentanaminium,hydroxide salt

A 10 mL round bottom flask, fitted with argon inlet and reflux condenserwas charged with 0.275 g(1.1 mM) ofN,N-dimethyl-N-[2-(methoxy)ethyl]-5,5,5-trifluoro-4-oxopentanaminium,hydroxide salt, 4.0 mL of water and 8.0 mL of 30% HBr in acetic acid.This mixture was heated at 120 C. for five hours, cooled, and thesolvent removed under reduced pressure. The residue was co-evaporatedwith 3×20 mL of water and the resulting oil was chromatographed on aDowex-1 (15×200 mm) column in the hydroxide form. Evaporation of theeffluent under reduced pressure gave 0.17 g(63%) of the product as thehydroxide salt. nmr(D₂ O)- 1.90(m,4H), 3.13(s,6H), 3.30(m,2H),3.48(m,2H), 4.01(bs,2H).

EXAMPLE IVN,N-dimethyl-N-[2-(phosphonooxy)ethyl]-5,5,5-trifluoro-4-oxopentanaminium,ammonium salt

A 10 mL three neck round bottom flask fitted with dropping funnel, argoninlet, magnetic stirrer, and ice salt bath is charged with 0.093 mL(1.0mM) of phosphorus oxychloride and 0.5 mL of trimethyl phosphate. To thismixture was added 70.0 mg(0.2 mM) ofN,N-dimethyl-N-[2-(hydroxy)ethyl]-5,5,5-trifluoro-4-oxopentanaminium,hydroxide salt dropwise at -10 C. The mixture was stirred for 30 minutesand then placed in a freezer overnight. The mixture was triturated withethyl ether followed by petroleum ether (4×50 mL) to give a resinousprecipitate. This precipitate was covered with ice, neutralized to pH6.0 with 1N NaOH, and evaporated to dryness under reduced pressure. Theresulting solid, 107 mg, was dissolved in 30 mM NH4OAc buffer at pH 7.0and purified by HPLC with system B. The product, which eluted at 12minutes, was isolated by lyophilization of the buffer to give 9.0mg(11%) of a colorless resin. nmr(D₂ O)- L 1.90(m,4H), 3.14(s,6H),3.42(m,2H), 3.60(m,2H), 4.19(bs,2H).

EXAMPLE V Half Sandwich Assay for Hexon Antigen from Adenovirus GeneralProcedure

Two-fold serial dilutions of purified Adenovirus hexon protein werecoated onto a polyvinyl microtiter plate in pH 9.5 carbonate buffer (10mM) for a period of 1-18 hours. The plate was rinsed several times witha solution consisting of 0.05% polyoxyethylenesorbitan monolaurate inphosphate buffered saline. The immunological reaction was performedusing an antibody (mouse-anti-hexon IgG) conjugated to alkalinephosphatase diluted to an appropriate level in 10% culture medium(containing fetal calf serum). After incubation for 1 hour, and astandard rinse procedure, a mixture of blocked modulator (10-3-10-4M)and RLE (10-8-10-9M) was added, -and the plate was preincubated for 10to 60 minutes. A chromogenic substrate (i.e. indoxyl butyrate oro-nitrophenyl butyrate) was added to the mixture. Color developmenttypically occured within 15 minutes, and could be stabilized for alonger period of time by the addition of excess free modulator. Wellswhich were coated with dilutions containing 1 ng/mL of hexon proteingave a positive response with this system.

EXAMPLE VI Sandwich Assay for Adenovirus

A polyvinyl chloride microtiter plate was coated with anit-adenovirusantibody by incubating the plate for 1 hour at 37° C. in a 1:900dilution of 4.5 mg/mL stock solution of the antibody in a 10 mMcarbonate 20 mM ethylenediaminetetraacetic acid (EDTA) buffer. The platewas washed 3 times with a wash buffer solution consisting of 0.5% caseinin 10 mM TRIS and 154 mM NaCl at pH 7.6. Adenovirus infected HeLa cellsdiluted to ca 1×10⁵ plaque forming units (PFU)/ml in pH 7.4 phosphatebuffered-saline containing 0.05% polyoxyethylenesorbitan monolaurate,0.1 mg/ML gentamicin, 0.5% phenol red, 0.1% bovine serum albumin, 10 mMEDTA and 100 mM ethylene bis(oxyethylenenitrilo) tetraacetic acid wereserially diluted in 2-fold steps across the plate. The plate wasincubated-as above to bind the cells to the antibody, washed with washbuffer, incubated with a 0.005 mg/mL tracer solution of anti-adenovirusconjugated to alkaline phosphatase in wash buffer containing 0.5%gelatin and 10% inactivated fetal calf serum. Excess tracer solution wasdecanted and the plate was washed 3 times with TRIS buffer (withoutcasein). A mixture of a 1×10⁻⁴ M solution of the blocked inhibitor fromExample I and 5×10⁻⁹ M RLE in 50 mM TRIS buffer, pH 8.5, was added andthe plate was incubated at room temperature for 10 minutes.o-Nitrophenyl butyrate (1 mM) was then added to each well of the plate.

Control wells having no antigen typically showed color detectable withthe naked eye in ca 15 minutes, and addition of free inhibitor (productB from Example I) to the control wells inhibited color formation forlonger periods. Test wells having antigen dilutions remained colorlessfor periods of time in excess of 15 minutes.

The FIGURE shows the relationship of color formation to antigenconcentration as determined with a Beckman DU7 spectrophotometer 20minutes after chromogen addition. It is seen that the optical density(OD) of the solution in the control wells is substantially constant atabout 0.24, and that the OD of the solutions in the test wells isinversely proportional to antigen concentration and approaches thecontrol value at very low antigen concentrations.

In summary, the invention provides a method for detection ordetermination of a ligand present in a liquid sample at very low levels.After binding the ligand, antiligand and tracer, the bound phase isseparated and contacted with a second enzyme and a blocked modulatorwhereby a first enzyme component of the tracer removes the blockinggroup to provide a modulator for the second enzyme. The resultingmodulator affects conversion of a substrate to a product by the secondenzyme leading to a detectable signal. Detection of the signalestablishes the presence or absence of the ligand in the sample. Bymeasuring the magnitude of the signal, the concentration of the ligandmay be determined. The modulator and the second enzyme provide twoamplification stages whereby the signal is amplified by 10⁶ fold ormore, enabling naked eye detection of the signal in a time up to 100fold less than by conventional EIA.

What is claimed is:
 1. A blocked enzyme inhibitor selected from thegroup of fluoroketones consisting of the general formulas I, II, III andIV ##STR7## wherein R₁ is t-butyl or ##STR8## R₂ is lower alkyl of 1-6carbon atoms, Y and Z are independently H of F wherein at least one of Yand Z is F; m is 2 to 3; n is 1-3l and B is an enzymaticallyhydrolysable group selected from the group consisting of a glycosylgroup hydrolysable by a glycosidase, an acyl group of 2-4 carbon atomshydrolysable by an esterase and a phosphoric acid, ester or salt thereofof the structure ##STR9## hydrolysable by alkaline phosphatase.
 2. Ablocked enzyme inhibitor of the formula ##STR10## where Y and Z areindependently H or F wherein at least one of Y and Z are F; X is O or S;and n is 1-4.
 3. A blocked enzyme inhibitor of the formula ##STR11##wherein n is 1-4; m is 2-4; and X is O or S.